Failure of the lung endothelial barrier leads to pulmonary edema and acute lung injury (ALI). Mechanical ventilation contributes to ALI mortality through excessive ventilatory stretch (VS), causing ventilator-induced lung injury (VILI). In animal models of VILI, increased endothelial nitric oxide (NO) production contributes to pulmonary endothelial barrier dysfunction. NO activates endothelial soluble guanylyl cyclase (sGC), producing cGMP but the effect of endothelial cGMP on VS-induced endothelial barrier function is unknown. Our preliminary experiments in a mouse model of VILI suggest that VS activates endothelial sGC to increase cGMP. After VS onset, sGC inhibition or stimulation attenuates or exacerbates barrier dysfunction, respectively, suggesting an injurious role for cGMP. In contrast, sGC activation before VS is barrier protective. Injurious VS also increases cGMP-activated phosphodiesterase 2A (PDE2A) mRNA and protein expression. PDE2A inhibition is protective. Inhaled bacterial lipopolysaccharide (LPS) also increases PDE2A expression and causes injury that is attenuated by either sGC or PDE2A inhibition. The overall goal of this application is to determine the effect of sGC-mediated cGMP production on endothelial permeability in VILI and in the combination of VILI with LPS inhalation to model septic ALI. We hypothesize that injurious VS increases endothelial 1) cGMP from NO-stimulated sGC and 2) PDE2A expression through activation of p38 and MAP kinase-activated protein kinase 2 (MK2) resulting in accelerated hydrolysis of endothelial barrier protective cAMP. In Aim 1, we plan to determine the relationships between VS, sGC activation, cytokine production, MAPK activation, cAMP and changes in pulmonary endothelial permeability in isolated mouse lungs, anesthetized ventilated mice, and monolayers of mouse lung microvascular endothelial cells subjected to injurious cyclic stretch, LPS and/or cytokine exposure. These experiments will utilize the novel sGC stimulator BAY 41-2272 and sGCa1-/- mice to manipulate lung sGC activity. In Aim 2, we will determine the roles of PDE2A and 3A in the complex effects of cGMP on pulmonary endothelial barrier function in lungs and endothelial monolayers subjected to injurious stretch LPS or cytokines. PDE mRNA, protein expression and activity will be studied. Specific inhibitors, a PDE2A shRNAmiR adenovirus to decrease endothelial PDE2A expression and PDE3A-/- mice will be employed in these experiments. In Aim 3, we plan to determine the upstream pathways responsible for the increased expression of PDE2A in VILI. Specifically, we plan to examine the roles of TNFa, p38, MK2 and NF?B using specific inhibitors, siRNA, as well as TNFa -/-, TNFa Receptor1-/-, MK2-/-, and p50-/- mice to dissect the involvement of each signaling pathway. These experiments will explore a novel mechanism of lung endothelial barrier dysfunction mediated by the simultaneous stimulation of sGC/cGMP and PDE2A expression by injurious levels of VS.